Bottom Line:
Two SCX and four ERLIC gradients were compared in details, and one ERLIC gradient was found to perform the best, which identified 2929 proteins, 583 phosphorylation sites in 338 phosphoproteins and 722 N-glycosylation sites in 387 glycoproteins from rat kidney tissue.In addition, this strategy enables identification of unmodified and corresponding modified peptides (partial phosphorylation and N-glycosylation) from the same protein.Interestingly, partially modified proteins tend to occur on proteins involved in transport.

ABSTRACTProtein post-translational modifications (PTMs) are regulated separately from protein expression levels. Thus, simultaneous characterization of the proteome and its PTMs is pivotal to an understanding of protein regulation, function and activity. However, concurrent analysis of the proteome and its PTMs by mass spectrometry is a challenging task because the peptides bearing PTMs are present in sub-stoichiometric amounts and their ionization is often suppressed by unmodified peptides of high abundance. We describe here a method for concurrent analysis of phosphopeptides, glycopeptides and unmodified peptides in a tryptic digest of rat kidney tissue with a sequence of ERLIC and RP-LC-MS/MS in a single experimental run, thereby avoiding inter-experimental variation. Optimization of loading solvents and elution gradients permitted ERLIC to be performed with totally volatile solvents. Two SCX and four ERLIC gradients were compared in details, and one ERLIC gradient was found to perform the best, which identified 2929 proteins, 583 phosphorylation sites in 338 phosphoproteins and 722 N-glycosylation sites in 387 glycoproteins from rat kidney tissue. Two hundred low-abundance proteins with important functions were identified only from the glyco- or phospho-subproteomes, reflecting the importance of the enrichment and separation of modified peptides by ERLIC. In addition, this strategy enables identification of unmodified and corresponding modified peptides (partial phosphorylation and N-glycosylation) from the same protein. Interestingly, partially modified proteins tend to occur on proteins involved in transport. Moreover, some membrane or extracellular proteins, such as versican core protein and fibronectin, were found to have both phosphorylation and N-glycosylation, which may permit an assessment of the potential for cross talk between these two vital PTMs and their roles in regulation.

pone-0016884-g001: Chromatograms and peptides distribution.SCX (A) and ERLIC (B) chromatograms of rat kidney tryptic peptides and distribution of total peptides (C), phosphopeptides (D), and glycopeptides (E). The scales of the Y-axes are different because 214 nm was monitored in (A) and 280 nm in (B).

Mentions:
As shown in Figure 1A and 1B, SCX and ERLIC fractionations generate completely different chromatograms due to their different separation principles. When SCX fractionation is conducted at pH 2.7, most of the tryptic peptides carry a net charge of +2 due to the positive charge at the C-terminal arginine/lysine and at their N-terminus [35]. Because of the negative charge from a phosphate group or sialic acid, most mono-phosphorylated peptides and mono-sialylated glycopeptides have a net charge of +1 and so are less well-retained by SCX materials and elute before most unmodified peptides [17]. Most multi-phosphorylated peptides and multi-sialylated glycopeptides are neutral or negatively charged and elute even earlier, frequently in the flow-through. Thus, unmodified peptides are separated from phosphopeptides and sialylated glycopeptides to a significant extent, and concurrent analysis of proteome, phosphoproteome and glycoproteome is potentially achieved in one run (Figure 1C-1E). Practically speaking, this approach is not completely successful. Peptides eluted in the flow-through are difficult to identify without further fractionation. Also, only about 30% of the phosphopeptides in a complex digest have a net charge of +1 or less at pH 2.7. The rest are distributed throughout the SCX gradient and so a second enrichment step such as titania or IMAC affinity chromatography is necessary to achieve good phosphopeptide identification.

pone-0016884-g001: Chromatograms and peptides distribution.SCX (A) and ERLIC (B) chromatograms of rat kidney tryptic peptides and distribution of total peptides (C), phosphopeptides (D), and glycopeptides (E). The scales of the Y-axes are different because 214 nm was monitored in (A) and 280 nm in (B).

Mentions:
As shown in Figure 1A and 1B, SCX and ERLIC fractionations generate completely different chromatograms due to their different separation principles. When SCX fractionation is conducted at pH 2.7, most of the tryptic peptides carry a net charge of +2 due to the positive charge at the C-terminal arginine/lysine and at their N-terminus [35]. Because of the negative charge from a phosphate group or sialic acid, most mono-phosphorylated peptides and mono-sialylated glycopeptides have a net charge of +1 and so are less well-retained by SCX materials and elute before most unmodified peptides [17]. Most multi-phosphorylated peptides and multi-sialylated glycopeptides are neutral or negatively charged and elute even earlier, frequently in the flow-through. Thus, unmodified peptides are separated from phosphopeptides and sialylated glycopeptides to a significant extent, and concurrent analysis of proteome, phosphoproteome and glycoproteome is potentially achieved in one run (Figure 1C-1E). Practically speaking, this approach is not completely successful. Peptides eluted in the flow-through are difficult to identify without further fractionation. Also, only about 30% of the phosphopeptides in a complex digest have a net charge of +1 or less at pH 2.7. The rest are distributed throughout the SCX gradient and so a second enrichment step such as titania or IMAC affinity chromatography is necessary to achieve good phosphopeptide identification.

Bottom Line:
Two SCX and four ERLIC gradients were compared in details, and one ERLIC gradient was found to perform the best, which identified 2929 proteins, 583 phosphorylation sites in 338 phosphoproteins and 722 N-glycosylation sites in 387 glycoproteins from rat kidney tissue.In addition, this strategy enables identification of unmodified and corresponding modified peptides (partial phosphorylation and N-glycosylation) from the same protein.Interestingly, partially modified proteins tend to occur on proteins involved in transport.

ABSTRACTProtein post-translational modifications (PTMs) are regulated separately from protein expression levels. Thus, simultaneous characterization of the proteome and its PTMs is pivotal to an understanding of protein regulation, function and activity. However, concurrent analysis of the proteome and its PTMs by mass spectrometry is a challenging task because the peptides bearing PTMs are present in sub-stoichiometric amounts and their ionization is often suppressed by unmodified peptides of high abundance. We describe here a method for concurrent analysis of phosphopeptides, glycopeptides and unmodified peptides in a tryptic digest of rat kidney tissue with a sequence of ERLIC and RP-LC-MS/MS in a single experimental run, thereby avoiding inter-experimental variation. Optimization of loading solvents and elution gradients permitted ERLIC to be performed with totally volatile solvents. Two SCX and four ERLIC gradients were compared in details, and one ERLIC gradient was found to perform the best, which identified 2929 proteins, 583 phosphorylation sites in 338 phosphoproteins and 722 N-glycosylation sites in 387 glycoproteins from rat kidney tissue. Two hundred low-abundance proteins with important functions were identified only from the glyco- or phospho-subproteomes, reflecting the importance of the enrichment and separation of modified peptides by ERLIC. In addition, this strategy enables identification of unmodified and corresponding modified peptides (partial phosphorylation and N-glycosylation) from the same protein. Interestingly, partially modified proteins tend to occur on proteins involved in transport. Moreover, some membrane or extracellular proteins, such as versican core protein and fibronectin, were found to have both phosphorylation and N-glycosylation, which may permit an assessment of the potential for cross talk between these two vital PTMs and their roles in regulation.